The immune response to insulin serves as a valuable model system for studying T cell recognition, antigen processing, and genetic regulation of immunity. Work done previously on this project lead to the discovery that disulfide reduction occurs during antigen processing and that T cells can recognize thiol-dependent epitopes. Disulfide reduction is both necessary and sufficient for binding of insulin peptides to class Il MHC and recognition by helper T cells. The mechanism responsible for disulfide reduction in the endocytic compartments of APC is unknown and will be investigated in specific aim 1, with the goal of identifying the intracellular compartments where reduction occurs and characterizing cofactors that may participate. Experiments with insulin B chain have demonstrated that peptides can bind to an alternative site in class Il MHC outside of the peptide-binding groove. The functional significance of alternative peptide/MHC complexes will be investigated in aim 2. Additional experiments in aim 2 will be done to characterize insulin T cell determinants with emphasis on the role of cysteines as MHC anchors or TCR contact residues. Specific aim 3 is to investigate a novel antigen processing pathway used by APC to present soluble insulin to Qa-1- restricted T cells, to study the peptide specificity of Qa-1 and specific T cells, and to explore the function of Qa-1-restricted T cells in the immune response to insulin. It is expected that the proposed study will broaden our general understanding of antigen processing, our knowledge of the peptide/MHC ligands that can be recognized by T cells, and the role of nonclassical class I molecules in immunity.